The present invention is a genetically-engineered animal such as a pig that is deficient in the .alpha. 1.fwdarw.3 galactosyl transferase gene, resulting in non-expression of galactosyl epitopes on its organs and tissues, or in masked expression of galactosyl epitopes on its organs and tissues, and methods for use thereof as a organ donor for humans.
Organ transplantation is now an increasingly successful option open to patients with end-stage disease of vital organs, but is limited by the availability of suitable donors. There is now a worldwide shortage of donor organs, and the number of potential recipients on waiting lists and the period of time that each waits for a suitable organ are both increasing annually, as reported in UNOS Annual Report (1990-1991) and UNOS Update 8, 1, 1992.
At the end of 1990, almost 22,000 patients awaited a solid organ transplant in the USA. One year later, the number had increased to over 25,000, despite the fact that approximately 15,000 organ transplants had been performed during this period. It is unlikely that the availability of human donors will ever be sufficient to match the rapidly increasing number of potential recipients.
One solution to the problem of organ supply would be the use of organs taken from a suitable animal donor. Although the higher non-human primates (apes and monkeys) would provide the closest immunological match for man, there are several factors that make the routine use of these species as organ donors unlikely. These include (i) inadequate numbers, (ii) difficulty and expense of breeding in large numbers, (iii) inadequate size of some organs (e.g., heart) for adult humans, (iv) probability of public concern regarding the use of such species for this purpose, and (v) risk of transfer of serious viral disease.
Attention is, therefore, being directed towards more commonly available mammals that are lower on the phylogenetic scale, in particular, the pig, which has many advantages in this respect, as reported by Kirkman, R. L. Of swine and men: organ physiology in different species. In Hardy, M. D. (ed), Xenograft 25, (Elsevier, Amsterdam, New York, Oxford, 1989), pp. 125-132, Cooper, D. K. C., et al. The pig as potential organ donor for man. In xenotransplantation. Cooper, K. D. C., Kemp, E., Reemtsma, K., White, D. J. G. (eds.) (Springer, Heidelberg, 1991), pp. 481-500. These include (i) availability in large numbers, (ii) inexpensive to breed and maintain, (iii) suitable size for the smallest or largest of humans, (iv) availability of pathogen-free (gnotobiotic) animals, and (v) considerable similarities of anatomy and physiology with man.
Survival of pig-to-man (or other primate) organ transplants is currently limited, however, by a severe humoral immune response that leads to destruction of the graft within minutes or hours, as reviewed by Cooper, et al. Experience with clinical heart xenotransplantation. In Xenotransplantation. Cooper, D. K. C., Kemp, E., Reemtsma, K., White, D. J. G. (eds.). (Springer, Heidelberg, 1991), pp. 541-557, and Cooper, et al. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J. Heart Transplant 7:238-246, 1988. The length of the period of survival of organ xenografts decreases with the increase of phylogenetic distance between donor and recipient species. Xenotransplants between closely-related species can usually survive the initial period of blood perfusion without damage, as do allotransplants. Subsequently, the foreign antigens of the transplanted organ trigger the recipient's immune response and the acute cellular rejection process begins. These xenografts, which behave clinically and histologically like allografts, are termed concordant xenotransplants. Xenografts between phylogenetically distant species follow a clinical course quite different from allotransplants and are termed discordant xenotransplants.
In discordant xenografted organs, vascular rejection occurs within a few minutes of recirculation, with a typical histopathological pattern of endothelial lesions with severe interstitial hemorrhage. This hyperacute rejection is usually irreversible, but can be delayed by removal of the recipient's natural antibodies against the donor tissue. There is now considerable evidence to suggest that this hyperacute rejection is entirely or largely a result of antibody-mediated complement activation through the classical pathway, as reported by Paul, L. C. Mechanism of humoral xenograft rejection. In Xenotransplantation. Cooper, D. K. C., Kemp, E., Reemtsma, K., White, D. J. G. (eds.) (Springer, Heidelberg, 1991), pp. 47-67, and Platt, et al. Mechanism of tissue injury in hyperacute xenograft rejection. In Xenotransplantation, pp. 69-79, and much attention is being directed towards inhibiting this humoral response.
A similar situation exists with regard to organ allografting across the ABO blood group barrier, from which much of the available information on antibody-mediated hyperacute rejection has been derived, as reviewed by Cooper, D. K. C. A clinical survey of cardiac transplantation between ABO-blood group incompatible recipients and donors. J. Heart Transplant 9:376-381, 1990, and Alexandre, et al., Present experiences in a series of 26 ABO-incompatible living donor renal allografts. Transplant Proc. 19:4538, 1987. The utilization of synthetic A and/or B blood group trisaccharides (Lemieux, R. U. Human blood groups and carbohydrate chemistry. Chem. Soc. Rev. 7:423-, 1978), covalently attached to a solid support in the form of an immunoadsorbent for the extracorporeal depletion of human anti-A and anti-B antibodies, has been shown to facilitate bone marrow and kidney transplantation across the ABO blood group barrier, as shown by Bensinger, et al. ABO-incompatible marrow transplants. Transplantation 33:427-429, 1982, and Bannett, et al., Experiences with known ABO-mismatched renal transplants. Transplant Proc. 19:4543-4546, 1987, respectively. Prolonged allograft survival even after the return of high titers of anti-A or anti-B antibody, and in the presence of normal levels of complement, has been documented by Alexandre and Bannett, supra, and has subsequently been termed "accommodation" by Bach, et al. Accommodation--the role of natural antibody and complement in discordant xenograft rejection. In Xenotransplantation, Cooper, D. K. C., Kemp, E., Reemtsma, K., White, D. J. G. (eds.), Springer, Heidelberg, 1991, pp. 81-99. Using similar methods, shorter periods of accommodation have also been documented following pig-to-baboon heart and kidney xenografting, as reported by Cooper, et al. Effects of cyclosporine and antibody adsorption on pig cardiac xenograft survival in the baboon. J. Heart Transplant 7:238, 1988, and Alexandre, et al., Plasmapheresis and splenectomy in experimental renal xenotransplantation. In: Hardy, M. D. (Ed.) Xenograft 25. (New York, Elsevier Science Publishers, 1989), p. 259.
An injectable form of the synthetic A and B blood group trisaccharides for the in situ "neutralization" of anti-A and anti-B antibodies (as originally investigated by Romano et al. Preliminary human study of synthetic trisaccharide representing blood substance A Transplant Proc. 19:4475-4478, 1987), has been demonstrated to prevent antibody-mediated hyperacute rejection in the baboon and, when combined with standard pharmacologic immunosuppressive therapy, extend experimental ABO-incompatible cardiac allograft survival from a mean of 19 minutes to more than 28 days, with one heart still functioning at almost 2 months, as reported by Cooper, et al., A novel approach to "neutralization" of preformed antibodies: cardiac allotransplantation across the ABO blood group barrier as a paradigm of discordant transplantation. Transplant Proc. 24:566-571, 1992.
However, it is clearly impractical to continually infuse the synthetic trisaccharides, or antibodies to the trisaccharides, into a patient, along with the immunosuppressive therapy, over an extended period of time.
As reported in the New York Times Feb. 3, 1993, The DNX Corporation is developing a pig with genes that are intended to mask the immunological markers present in pigs that are used as a source of donor organs for implantation into humans. These pigs are created by microinjection of human DNA into pig embryos. However, the end result is not that the pig genes are eliminated, but that the cells also express human markers.
It is therefore an object of the present invention to provide a long term solution to the problem of alleviating immunorejection of xenotransplants, specifically pig into human, where the rejection is mediated by the glycoprotein structures present on the xenotransplant which are not found in the human.
It is a further object of the present invention to provide genetically engineered tissues which do not express sugars which may elicit an immune, especially a complement-mediated, response following transplantation of an animal organ into a human.